PROJECT SUMMARY/ABSTRACT Pediatric arterial ischemic stroke afflicts ?2,000 U.S. children every year, permanently disabling most, yet is poorly understood. The Vascular effects of Infection in Pediatric Stroke (VIPS I) study established an international network of 37 sites that enrolled 355 children with stroke and 354 controls. We discovered: (a) minor clinical infections act as a stroke trigger, while routine childhood vaccinations are protective; (b) almost half of children with stroke have an acute herpesvirus infection (the cause of chicken pox, cold sores, and other common illnesses); and (c) children with stroke have a high risk of recurrent stroke, particularly if they have an arteriopathy. A VIPS pilot study suggests that other common childhood pathogens may also play a role, possibly in combination with herpesviruses. The full spectrum of pathogens that contribute to childhood stroke risk remains unknown. Infection is compelling as a treatable stroke risk factor, with available therapies for both pathogens and downstream inflammatory effects. However, VIPS I findings present a paradox: infection is common, while childhood stroke is uncommon. Possible explanations are (1) the ?infection hypothesis?: unusual pathogen strains, or combinations of pathogens, lead to stroke; and (2) the ?host response hypothesis?: an unusual inflammatory response to infection leads to stroke. A thorough understanding of both the specific pathogens and inflammatory mediators underlying childhood stroke pathogenesis, particularly arteriopathic stroke, is needed to develop strategies to reduce recurrent stroke in children. Broad testing for infections through traditional methods is prohibitively costly, and requires preconceived assumptions about the pathogens involved. Next generation sequencing (NGS) allows for the efficient, highly sensitive and unbiased detection of any pathogen, known or novel, by sequencing all nucleic acids in a biological sample and matching them with genomes in a reference database. To study the inflammatory response, multiplex bead arrays can now efficiently test for a large number of immune mediators on tiny blood samples. VIPS II will use these cutting-edge techniques to address the VIPS I paradox and better understand the causes of childhood stroke. The specific aims are to (1) identify known and novel pathogens in children with stroke, and determine whether different pathogens are seen with arteriopathic versus other stroke types; (2) determine whether children with arteriopathic stroke have a different inflammatory response compared to those with other stroke types; and (3) use data from Aims 1 and 2 to explore different mechanistic pathways from infection, to inflammation, to arteriopathic stroke, and other stroke types. The goal of VIPS II is to gain the knowledge needed to protect children with stroke from additional brain injury. Its results will guide the selection of currently available therapies?such as antimicrobials and anti- inflammatory medications?for a pediatric clinical trial aimed at preventing recurrent stroke.